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標題: The Expression of Glutathione S-Transferase GST-3 in Susceptible and Resistant Diamondback Moth and its Overexpression in E.coli
作者: Huang, Huey-shiang
關鍵字: 小菜蟲;麩胱甘月太轉酉每
出版社: 昆蟲學研究所
在十字花科蔬菜害蟲小菜蛾 Plutella xylostella (L.)幼蟲體內,已知有四個glutathione S-transferase(GST)的同功異構酉每。其中兩個生化及毒理相近的異構酉每GST-3及GST-4,和此蟲對一些殺蟲劑抗藥性有關。
本研究利用已選殖的GST-3 cDNA (809 bp)為探針,偵測感性與抗性小菜蛾幼蟲體內GST-3 mRNA的量以及genomic DNA中GST-3基因的數目。另外,並利用E. coli表現GST-3 cDNA所encode的蛋白質,測定其生化及毒理性狀。
小菜蛾幼蟲的RNA,經電泳及轉印到nylon membrane上,和GST-3 cDNA及小菜蛾actin部分cDNA(200 bp)進行雜合反應。雜合的結果顯示抗性小菜蛾幼蟲體內GST-3 mRNA的量多於感性小菜蛾,所雜合到的transcript估計有1.lkb。將感性及抗性小菜蛾之genomic DNA以三種限制酉每Cla I、Hind III、Nco I剪切後,與GST-3 cDNA進行雜合,結果顯示感性及抗性小菜峨之GST-3基因套數並無明顯不同,因此兩者GST-3 mRNA量的差異並不是由gene amplification造成。
將GST-3 cDNA構築在表現載體pET-21b內,送入 E. coli BL21品系中。所表現的蛋白質經affinity column純化後,測定其subnit molecular mass、pI、對GST-3 antiserum的交互作用,以及對一些受質的 conjugation,確定與小菜蛾幼蟲體內純化之GST-3相同。

Among the four glutathione S-transferase (GST) isozymes characterized from diamondback moth (DBM), Plutella xylostella, two immuno-related, GST-3 and GST-4, are involved in this insect resistance to some insecticides. A 809 bp cDNA of GST-3 cloned earlier was used as a probe in the present study to detect the mRNA and the copy number of GST-3 gene in a susceptible and a methyl parathion-resistant (MPA) strains of DBM. Considerably more GST-3 transcript of ca.1.1 kb was observed in the MPA than in the susceptible strains, in consistence with the amount of the GST-3 proteins detected in these two strains. Southern blot of genomic DNA digested with Cla I, Hind III or Nco I indicated that there was no difference in copy number of GST-3 gene in these two strains. These results suggest that the elevated expression of GST-3 in MPA strain was not due to gene amplification. The coding region of the GST-3 cDNA was cloned into pET-21b and expressed in E. coli. The protein purified with affinity chromatography from the bacterial culture and the GST-3 isolated from DBM appeared to have similar biochemical and toxicological properties.
Appears in Collections:昆蟲學系

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